Ubiquitin (Ub) is a 76 amino acid protein. Many biochemical pathways are regulated in part by post-translational modification of proteins with Ub and ubiquitin-like (UbL) molecules. This post-translational protein modification by Ub, a process known as ubiquitination or ubiquitylation, is involved in the regulation of biological processes such as protein degradation, gene transcription, cell-cycle progression, DNA repair, apoptosis, virus budding and receptor endocytosis. Precise regulation is achieved through the opposing actions of Ub/UbL-specific conjugating and deconjugating enzymes. Members of the Ub and UbL protein family include Ub, SUMO, NEDD8, ISG15, URM1, FAT10, UFM1, LC3, GATE-16, GABARAP and ATG12. These related proteins are structurally similar, and are activated, conjugated, and released from conjugates in a mechanism akin to that for Ub. There is cross-talk between conjugation pathways with some substrate proteins becoming targeted by more than one type of modifier. Ub is found only in eukaryotic organisms in which it shows strong sequence conservation.
Post-translational modification of proteins by Ub and UbLs regulates almost every aspect of biology and enables complex and reversible regulation of protein stability and activity. Because of this broad role in cell biology, dysfunction in Ub pathway enzymes results in a multitude of human diseases, and suggests there is a large population of enzymes that may be amenable to small molecule manipulation. Covalent attachment of Ub/UbLs to substrates is achieved through a canonical E1-E2-E3 enzyme cascade, whereby Ub is activated by E1 and transferred to an E2 via a high-energy thioester bond. E2 carrying activated Ub then binds to an E3 enzyme, where Ub transfer to a lysine residue on substrate is poised to occur. The mechanics of transfer from E3 to substrate depends on the specific class of E3 ligase involved. These ligases comprise over 500 different proteins and are categorized into multiple classes defined by the structural element of their E3 functional activity. The E3 ligases are grouped into two main classes: the HECT ligases containing an active site cysteine which serves to accept Ub prior to substrate transfer, and the RING E3 ligases, which contain zinc finger-like domains that act as scaffolds enabling transfer of Ub directly from an E2 enzyme to a substrate. The RBR (RING-between-RING) is a subclass of E3 Ub ligases that are considered RING/HECT hybrids in that similar to RING ligases coordinate zinc and bind E2, but also contain an active site cysteine similar to HECT ligases. Specifically, both HECT and RING ligases transfer an activated Ub from a thioester to the 8-amino acid group of a lysine residue on a substrate; however, HECT ligases have an active site cysteine that forms an intermediate thioester bond with Ub, while RING ligases function as a scaffold to allow direct Ub transfer from the E2 to substrate.
Parkin is a RBR E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson's disease, cancer and mycobacterium infection. The RBR family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed RING/HECT hybrid enzymes. Parkin has been proposed to function as a RBR ligase such that it encompasses both of the major classes of E3 ligase in one protein. Specifically, it may function with both a catalytic cysteine and a classical RING motif for binding E2. While recent work has established that Parkin has four RING domains, coordinating eight zinc (Zn) molecules, the exact residues coordinating these Zn atoms, and the organization of each of the RING domains with respect to each other are not known. Parkin has been described to have latent activity that can be activated with carbonyl cyanide 3-chlorphenylhydrazone (CCCP) in cells, although it is not completely known how the latent state becomes activated at the molecular level, and whether purified Parkin protein contains a similar latent state has not yet been established. Regulation of Parkin activity by phosphorylation has been described, but the subsequent molecular events post-phosphorylation are not understood. Finally, while catalytic networks have been investigated for E3 ligases it is not yet clear whether they function with a classic triad/dyad-based mechanism, or whether catalysis occurs through a hydrogen-bonding network. For deubiquitinating enzymes (DUBS) it has been demonstrated that the cleavage of Ub from a substrate occurs through a classic triad/dyad mechanism, utilizing a critical catalytic cysteine residue, and a histidine residue in close proximity.
The process by which Ub is added to a protein by an E3 Ub ligase is the reverse reaction of removing Ub executed by deubiqutinating enzymes (DUBs). A variety of Ub activity probes have been developed to monitor the removal of Ub by DUBs. As described herein, Ub activity probes can also be used to screen compounds screen to identify small molecules that activate or inhibit E3 Ub ligases.